从美国癌症协会的研究学者资助（116403-RSG中-09-082-01-强制性）和广深高速壁间的维克森林大学健康科学基金，支持这项工作的一部分。星展得到了NCI的培训补助金5T32CA079448。

1。生成的shRNA构造<ol><li>确定目标的siRNA序列（20-23核苷酸）根据先前公布的标准，或使用一个基于网络的算法服务器，如“siRNA的目标搜索”从Ambion公司和金斯瑞。 </li><li>根据表1，图1和例子序列设计合成寡核苷酸。进行PCR扩增使用的寡核苷酸P1和P2（30个循环变性94°为30秒，退火60°为30秒，30秒72°C时的伸长）和质粒携带U6或H1子作为一个模板。用PCR纯化柱纯化PCR产物。经BamHI酶切消化和净化消化的DNA，用PCR纯化柱。退火的寡核苷酸P3和P4（2.5 pmol /每微升100μL的50 mM Tris盐酸，pH值8.0），煮沸5分钟，慢慢冷静下来，以环境温度。消化慢病毒VEC器，如一个描述在图2A，经BamHI和EcoRI，执行凝胶净化。 </li><li>开展BamHI酶切/酶切消化的载体，的BamHI /酶切消化PCR片段和一个退火寡核苷酸对（形成HindIII和EcoRI位点在两端）一时10分10秒的摩尔比在1 3个片段结扎（ 图1）。 </li><li>变换高效的主管大肠杆菌大肠杆菌 DH5α的细胞使用结扎产品。筛选菌落使用寡核苷酸小五（载体）和P6（H1或U6启动子）7。 </li><li>准备从商业套件的阳性克隆质粒DNA，并确认存在插入的BamHI / EcoRI酶切和DNA琼脂糖凝胶电泳。包含使用寡核苷酸小五子和shRNA序列的区域。 </li><li>使用MIDI-或Maxi准备质粒DNA试剂盒（内毒素免费优先），准备携带shRNA表达的慢病毒DNA录像带。确定DNA浓度测量在260 nm处的吸收。检查DNA的纯度，使用260 nm/280 nm的比例，并确保它在1.8至2.0范围内。检查的慢病毒载体的完整性，使用琼脂糖凝胶电泳。转染的DNA应该是超螺旋。 </li></ol> 2。慢病毒转染 注意事项 ：慢病毒载体是由人类免疫缺陷病毒1（HIV-1）基因组和复制的无能而得。他们已被广泛用于基因传递因感染分裂和非分裂细胞的能力。然而，存在两个主要的风险在使用慢病毒的研究。 （1）潜在的复制能力慢的一代。这种风险可以大大降低，如果使用第三代慢病毒系统。 （2）潜在的癌变。如果进行插入致癌或抑制肿瘤抑制基因，可以加剧这种风险。基于艾滋病毒，慢病毒有关的研究活动应当遵循“慢病毒载体的研究与生物安全注意事项”的美国国立卫生研究院（ <a href="http://oba.od.nih.gov/rdna_rac/rac_guidance_lentivirus.html" target="_blank">http://oba.od.nih.gov/rdna_rac/rac_guidance_lentivirus.html</a> ），并要求从生物安全委员会的批准研究员的机构。一般情况下，加强生物安全2级（BSL-2）遏制需要实验室设置，如果使用慢病毒载体。 <ol><li>板4×10 6的HEK 293T细胞完整的DMEM培养基（提供10％胎牛血清（FBS），2毫米L-谷氨酰胺，100个单位/ ml青霉素，100μg/ mL链霉素的DMEM培养液）在10厘米的菜肴和文化隔夜在37°C 5％CO2培养箱。取代我降低血清培养基（Invitrogen公司）5毫升预热的OPTI-MEM培养基。 </li><li>准备溶液A：10微克的shRNA慢病毒载体，5微克每第三根儿ATION慢病毒包装质粒（制备高纯度;三种包装质粒VSV-G的包膜蛋白，PRSV-REV： 转速 ，pMDLg / pRRE：GAG / POL，这是必不可少的慢病毒包装），36 2，2M氯化钙和300μl无菌水的微升。 </li><li>准备B液：2×HEPES缓冲液300μL（281 mM氯化钠，100毫米，1.5毫米的Na 2 HPO 4，酸碱度为0.5 N氢氧化钠调整至7.12和0.22微米过滤消毒）肝素钠。 </li><li>慢慢下降到溶液B的解决方案，而冒泡通过溶液B用吸管的空气。离开管在环境温度为30分钟。 </li><li>慢慢下降到步骤2.1准备的HEK 293T细胞培养菜的混合溶液A / B和在5％CO 2培养箱孵育37°C时为4-6小时。 </li><li> 7毫升完整的DMEM培养基更换介质。 24小时后，一个完整的DMEM培养基添加5毫升。收获的中等续aining 24小时后，另一个文化的慢。 </li><li>在1500 rpm时，环境温度为10分钟旋转介质。使用0.45微米的过滤器过滤上清液，旋转，通过流动介质中的含超速25000转的慢（等于125000×G与SW28摆动转头，贝克曼超速离心机XL-90），4℃时为90分钟ç。上清倒出到容器中，用5％的漂白剂（终浓度）。 </li><li>慢病毒颗粒和悬浮在0.5-1.0毫升的冷PBS慢等分，每管到50-100微升。储存在-80°C。 </li><li>慢病毒滴度可以由商业套件（如QuickTiter慢病毒从细胞Biolabs公司的定量试剂盒，公司），实时PCR协议8，或通过确定共同表达的荧光标记，用荧光显微镜或流式细胞仪。通常，一个10厘米的培养皿中可产生约0.5〜1.0×10 8感染带病单位（IU）的。 </li></ol> 3。感染细胞的感染和表征<ol><li>种子在30-40％汇合（约5-8×10 5细胞，根据细胞大小）2毫升完全培养基在6孔板被感染的细胞。文化细胞一夜之间在37°C 5％CO2培养箱。 </li><li>更换新鲜培养液1毫升含8微克/毫升的聚凝胺（Sigma公司，猫＃H9268）或5微克/毫升鱼精蛋白（Sigma公司，猫＃P4020）介质。 </li><li>确定一个合适的多重感染（MOI），范围为一个特定的细胞用荧光标记的慢线9。计算或凭经验确定被用来感染细胞的慢病毒量。慢慢下降到板慢，轻轻摇晃10秒。 </li><li>在37℃孵育板在5％ 二氧化碳培养箱6小时，然后替换正常的完全培养基介质。 </li><li>至少有2大YS感染后，检查细胞，荧光显微镜使用慢病毒表达的荧光标记物和/或添加相应的抗生素，以表达抗生素抗性基因的慢病毒载体的媒介。为诱导载体，如在春节系统（ 图2），文化与四环素无胎牛血清培养基中的细胞准备。分裂细胞感染后2-3天。采取部分细胞培养介质中提供1.5微克/毫升≥2天，强力霉素（DOX）和无诱导的基因敲除测试。 </li><li>检查印迹，实时PCR或免疫组化感染后≥2天，2天之后，抗生素的选择，或≥2天之后，DOX除了为春节上诱导系统的基因敲除。 </li><li>利用不同的方法（如细胞增殖实验，软琼脂文化研究，迁移检测，入侵检测和肿瘤形成的研究），以确定目标基因KN的影响细胞恶性肿瘤ockdown上。 </li></ol> 4。移植瘤小鼠模型的研究<ol><li>清洁鼠标麻醉和70％乙醇注射，以防止感染小鼠的所有表面。麻醉裸鼠裸鼠（NCI弗雷德里克）使用2％异氟醚在麻醉诱导室细胞接种前10分钟的氧气混合。维持麻醉用1％异氟醚混合氧气通过鼻锥。在通风良好的房间进行这一步，异氟醚清道夫过滤器附加到感应室。 </li><li>传播携带的shRNA的细胞。春节，在诱导载体使用四环素的培养基。 Trypsinize细胞和悬浮在含有50％的基底膜完整的培养基。放置在加热垫的裸鼠（30℃）和200μL细胞（1-10×10 6，取决于细胞恶性肿瘤）注入小鼠，小鼠注射部位用1-2％。使用注射器25。5号针头的皮下注射，原位注射28-30表针。 </li><li>对于构成的shRNA载体，利用2组小鼠注射细胞与靶基因的shRNA和一个加扰的shRNA表达。对四环素诱导的shRNA载体，利用4组，携带靶基因的shRNA和一个加扰的shRNA，与正常的水供应和阿霉素（1.5毫克/毫升）含有水（2的shRNA×2治疗= 4组）的细胞注入小鼠。每周两次更换DOX含有水。 </li><li>监测的肿瘤每周或双周数字游标卡尺的长度和宽度，并计算肿瘤体积公式V = 1/2（长×宽2）10（五）。确保该肿瘤体积不超过的体制ACUC的指引（例如，体重的10％）。安乐死的任何鼠标使用由体制ACUC批准的协议，如果它显示任何广泛的溃疡或坏死，感染的明显迹象N，不受控制的出血或终末期疾病。 </li><li>接种肿瘤细胞表达生物发光标记11，如萤火虫荧光素酶可以监测肿瘤生长和转移。清洁鼠标麻醉和70％的乙醇，以防止感染小鼠的成像表面。麻醉小鼠步骤4.1中所述。注入荧光素（150毫克/公斤）腹腔。放入消毒成像室的商业成像系统的IVIS成像系统（卡尺生命科学公司），如小鼠。麻醉杆转动，以保持与氧混合的1％异氟醚麻醉。在通风良好的房间进行这一步。 </li><li>采取暴露5分钟的第一张图片，并检查其信号强度。调整曝光时间，以获得最佳的信号与背景图像。凭经验判断成像时间，一般是1-5分钟的皮下肿瘤。 </li><li>安乐死的小鼠，由批准的协议由体制ACUC时，肿瘤大小达到约1000毫米，或指定由ACUC指引。海关的异种移植肿瘤，称重，并拍照留念。处理不同的研究，病理分析，以确定肿瘤细胞的形态，如肿瘤样本，Western blot和实时PCR研究测试基因表达。 </li></ol> 5。代表结果该协议被用来研究萤火虫荧光素酶表达的MDA-MB-231细胞（人乳腺腺癌细胞;卡尺生命科学）异种移植肿瘤的形成，影响YY1击倒在裸鼠裸鼠的影响。对人类的影响YY1的shRNA靶序列“GGG AGC的AGA公司AGC AGG的炉TGC AGA T”型。一个加扰序列“GGG ACT ACT CTA的TTA总工会的CAT牛逼”也创造了控制（续）shRNA的，没有任何已知的转录显着相似。用于春节，诱导的shRNA结构的寡核苷酸与影响YY1，作为一个例子，如表1所示。作为一个结果，PLU-PURO指数上涨的影响YY1 shRNA和PLU-PURO INDU-CONT的shRNA慢病毒载体，构建了它们被用来生产慢病毒。我们利用这些慢病毒单独感染两个MDA-MB-231细胞克隆（克隆1和3），同时表达四环素调节（四环素）和萤火虫荧光素酶。嘌呤霉素筛选后，获得多克隆细胞群。 图3A显示阿霉素诱导的影响YY1 MDA-MB-231细胞在由PLU PURO-INDU影响YY1的shRNA慢病毒感染的这些击倒。然后，我们使用单独通过这些诱导影响YY1的shRNA和cont的shRNA慢病毒感染，并指出，影响YY1消耗降低MDA-MB-231细胞的侵袭（ 图3B）多克隆细胞克隆3。 Western blot分析证实DOX诱导的MDA-MB-231与INDU影响YY1的shRNA细胞的影响YY1沉默，而含有INDU-CONT的shRNA的细胞没有表现出这种效果（ 图3C）。然后，我们使用<html>这些细胞移植瘤小鼠模型的研究。 INDU-shRNA的影响YY1与MDA-MB-231细胞植入和DOX含水提供的小鼠与对照组相比，显示减少肿瘤的形成，当发光（ 图4A）可视化，并确定肿瘤的重量（ 图4B ）。在这些移植瘤的影响YY1沉默如图4C所示用Western blot研究证实。 <img alt="图1" src="/files/ftp_upload/4129/4129fig1.jpg" /> 图1。示意图产生一个shRNA的结构和shRNA转录引物P1到P6（例如序列表1）所示。波尔三：RNA聚合酶III（D）：消化结束。图纸是不是规模。 <a href="http://www.jove.com/files/ftp_upload/4129/4129fig1large.jpg" target="_blank">点击这里查看大图</a> 。 2“SRC =”/ files/ftp_upload/4129/4129fig2.jpg“/&gt; 图2。示意图（A）的慢病毒载体（B）该机制的四环素诱导H1启动子用于基因沉默 。慢病毒载体重建基础上pLL3.7 14。 H1-PR：H1启动子; UBC-PR：人类泛素C启动; PURO：嘌呤;居民企业：四环素反应元件;霉素：强力霉素。四环素：四环素调节。 5&#39;LTR，3&#39;SIN-LTR和WRE的是一个慢病毒载体14的重要组成部分。 <img alt="图3" src="/files/ftp_upload/4129/4129fig3.jpg" /> 图3。阿霉素诱导的影响YY1沉默和它的MDA-MB-231细胞侵袭的影响 。 A.影响YY1水平在来自两个多克隆细胞人口四环素表达PLU PURO-INDU影响YY1的shRNA慢病毒感染和阿霉素在缺乏和存在的培养的克隆1和3。 B. Boyden小室检测的MDA-MB-231细胞诱导的shRNA。 （* P &lt;0 0与其他三组.05）。 C.代表西方影响YY1在这四个细胞群表达的印迹。 <img alt="图4" src="/files/ftp_upload/4129/4129fig4.jpg" /> 图4。对MDA-MB-231细胞移植瘤形成的影响YY1诱导沉默 。 A.细胞移植（左）和代表发光图像的IVIS成像系统捕获后的细胞接种在第4周（右）示意图。 B.异种移植在4个星期的肿瘤重量。 * p≤0.05。 C.印迹影响YY1和β-肌动蛋白INDU-shRNA的影响YY1阿霉素在缺乏和存在的MDA-MB-231细胞移植瘤中的表达。在上面的标签是单个小鼠的名字。 <table border="1"><tbody><tr><td> 寡核苷酸 </td><td> 序列（5&#39; - 3&#39;） </td><td> 用法和位置 </td></tr><tr><td> 小一 （春节的H1） </td><td> CAGT GGATCC 海巡署ACG的CTG的ACG的氯乙酸TCA的行政协调会ç </td><td>聚合酶链反应; H1启动子5&#39;-端</td></tr><tr><td> P1（U6的） </td><td> CAGT GGATCC GAC海湾合作委员会海湾合作委员会空中交通管制大老山隧道AGG的 </td><td>聚合酶链反应;在U6启动子的5&#39;-端</td></tr><tr><td> P2（影响YY1与春节，在H1） </td><td> CAGC AAGCTT棉酚ATC TGC ACC TGC TTC TGC TCCÇGGG 空管大老山隧道空中交通管制的ACT GAT的AGG的棉酚Ç </td><td>聚合酶链反应;在春节上诱导H1启动子的3&#39;-末端</td></tr><tr><td> P2（与U6的影响YY1） </td><td> CAGC AAGCTT棉酚ATC TGC ACC TGC TTC TGC TCCÇAAA CAA GGC TTT TCT CCA AGG GAT 1 </td><td>聚合酶链反应;在U6启动子的3&#39;-末端</td></tr><tr><td> 三 （的影响YY1） </td><td> AGC TT ATC TGC ACC TGC TTC TGC TCCÇTTTTTĞ </td><td>要退火与P4 </td></tr><tr><td> 小四 （对影响YY1） </td><td> aattc AAAAA <strONG&gt; GGG AGC AGA AGC AGG TGC AGA TA </td><td>要退火与P3 </td></tr><tr><td> 小五 （为pLL3.7） </td><td> TAC AGT GCA GGG GGG GAA AGA ATAĞ </td><td> PCR筛选，使用与P6 </td></tr><tr><td> P6（春节，在H1） </td><td> GAT的交委会CCA棉酚成飞阿拉木图图文影像交流</td><td>对于PCR筛选，与小五</td></tr><tr><td> 六 （U6的） </td><td> AGG的GTG艾格特交委会铁通TTG炉TGC甘油三酯</td><td>对于PCR筛选，与小五</td></tr></tbody></table> 表1。寡核苷酸合成中产生的shRNA结构。P1和小六为小鼠的U6和春节上诱导人H1启动子序列。作为人类影响YY1的shRNA靶序列与GGG AGC的AGA AGC的AGG的炉TGC AGA公司T.的序列，具体到影响YY1突出的例子。两个P2序列的影响YY1分别设计的两个促销员。构U6 /年Y1的shRNA的前面所述15日 ，而春节在H1/YY1在这个协议中使用。 P5是载体，具体为pLL3.7 14所示的顺序。限制性内切酶的序列强调，而序列的PCR扩增过程中退火发起人斜体。

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Pathol. , (2012).</li><li>Sui, G., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+silencing+by+a+DNA+vector-based+RNAi+technology.">Gene silencing by a DNA vector-based RNAi technology.</a> Methods in Molecular Biology. 309, 205-218 (2005).</li><li>Trower, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+PCR-based+colony+screening+protocol+for+cloned+inserts.">A rapid PCR-based colony screening protocol for cloned inserts.</a> Methods Mol. Biol. 58, 329-333 (1996).</li><li>Lizee, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quantitative+reverse+transcriptase-polymerase+chain+reaction+as+a+method+for+determining+lentiviral+vector+titers+and+measuring+transgene+expression.">Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression.</a> Hum. Gene Ther. 14, 497-507 (2003).</li><li>Zhang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significance+of+controlled+conditions+in+lentiviral+vector+titration+and+in+the+use+of+multiplicity+of+infection+(MOI)+for+predicting+gene+transfer+events.">The significance of controlled conditions in lentiviral vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer events.</a> Genet Vaccines Ther. 2, 6 (2004).</li><li>Geran, R. I., Greenberg, N. H., MacDonald, M. M., Schumacher, A. M., Abbott, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+for+screening+chemical+agents+and+natural+products+against+animal+tumors+and+other+biological+systems.">Protocols for screening chemical agents and natural products against animal tumors and other biological systems.</a> Cancer Chemother. Rep. 3, 1-103 (1972).</li><li>Massoud, T. F., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+in+living+subjects:+seeing+fundamental+biological+processes+in+a+new+light.">Molecular imaging in living subjects: seeing fundamental biological processes in a new light.</a> Genes Dev. 17, 545-580 (2003).</li><li>Inoue, H., Nojima, H., Okayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+transformation+of+Escherichia+coli+with+plasmids.">High efficiency transformation of Escherichia coli with plasmids.</a> Gene. 96, 23-28 (1990).</li><li>Toledo, J. R., Prieto, Y., Oramas, N., Sanchez, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylenimine-based+transfection+method+as+a+simple+and+effective+way+to+produce+recombinant+lentiviral+vectors.">Polyethylenimine-based transfection method as a simple and effective way to produce recombinant lentiviral vectors.</a> Appl. Biochem. Biotechnol. 157, 538-544 (2009).</li><li>Rubinson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lentivirus-based+system+to+functionally+silence+genes+in+primary+mammalian+cells,+stem+cells+and+transgenic+mice+by+RNA+interference.">A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference.</a> Nat Genet. 33, 401-406 (2003).</li><li>Sui, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yin+Yang+1+is+a+negative+regulator+of+p53.">Yin Yang 1 is a negative regulator of p53.</a> Cell. 117, 859-872 (2004).</li></ol>

没有利益冲突的声明。

本协议描述了一个方法来打倒使用shRNA基因和可视化的生物效应，利用生物发光成像在体内的肿瘤细胞的生长。一个shRNA的目标站点不应该包含任何三个酶切位点（的BamHI，HindIII和EcoRI位），用于亚克隆。在罕见的情况下，当任何这些网站是目前一个额外的限制性内切酶可以用来代替它产生的构造。 在这个协议中的几个关键步骤将加快shRNA慢病毒载体的建设。首先...

除了常见的RNA和蛋白质分析和细胞培养实验室设备，该协议需要以下材料和试剂。 <table border="1"><tbody><tr><td> 试剂名称 </td></tr><tr><td>生物安全柜适用于生物安全等级2围堵</td></tr><tr><td> PCR扩增热循环仪</td></tr><tr><td>超速离心机</td></tr><tr><td>麻醉诱导室与异氟醚拾荒者</td></tr><tr><td>数字游标卡尺</td></tr><tr><td>的IVIS成像系统</td></tr><tr><td>高主管DH5a大肠杆菌</td></tr><tr><td>酶切，BamHI酶切，酶切限制性内切酶</td></tr><tr><td> T4 DNA连接酶</td></tr><tr><td>第三代慢病毒包装质粒VSV-G的，PRSV牧师和pMDLg的的/ pRRE </td></tr></tbody></table> 材料清单

dna vector-based rna interference to study gene function in cancer

专门降解靶mRNA，RNA干扰（RNAi）抑制基因的表达。双链小干扰RNA（siRNA）在1基因沉默的发现以来，RNA干扰已成为强大的研究工具，在基因功能研究。 RNAi介导的基因沉默基因缺失相比，具有许多优点，如与它进行的易用性和适合大多数细胞株。多项研究已经证明，在癌症研究RNAi技术的应用。 DNA载体为基础的技术，以生产小发夹RNA（shRNA）由U6或H1启动驱动的发展，特别是长期和诱导的基因沉默可能2,3。其组合使用遗传工程病毒载体如慢，有利于高效率的shRNA交付和/或集成到稳定的shRNA表达的基因组DNA。 我们描述1 detaile的ð程序使用的基于DNA载体的RNAi技术，以确定基因的功能，包括建设慢病毒载体表达的shRNA慢病毒生产和细胞感染，并利用小鼠移植瘤模型的功能研究。 据报道，在各种战略已产生的shRNA结构。这里描述协议采用PCR扩增和3片段结扎，可以用来直接和有效地产生shRNA的含不留任何额外的核苷酸毗邻到的shRNA编码序列的慢病毒结构。由于磁带创建这一战略的shRNA表达限制性内切酶可以削减，他们可以很容易地转移到其他载体具有不同的荧光或抗生素标记。大多数商业化的转染试剂可用于生产慢。然而，在这份报告中，我们提供了一个经济的方法，使用磷酸钙沉淀，可以实现90％以上的转染效率293T细胞。相比构shRNA表达载体，诱导shRNA的系统特别适合撞倒必不可少的细胞增殖的基因。我们证明了阴阳1（YY1），潜在的癌基因在乳腺癌4,5基因沉默，一个春节的shRNA诱导系统和肿瘤形成的影响。使用慢病毒的研究需要由一个研究员的机构生物安全委员会审查和批准的生物安全议定书。利用动物模型的研究需要动物协议的动物护理和使用委员会研究员的机构（ACUC）审查和批准。

Watch this Scientific Journal Video about DNA Vector-based RNA Interference to Study Gene Function in Cancer at JoVE.com

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA干扰（RNAi）拥有超过基因敲除的许多优点，在基因功能研究的工具已被广泛使用。基于DNA载体的RNAi技术的发明，已作出长期和诱导基因敲除可能，也增加了基因沉默的可行性<em&gt;在体内</em&gt;。

基于DNA载体的RNA干扰在癌症研究基因功能

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

dna-vector-based-rna-interference-to-study-gene-function-in-cancer

Research

JoVE Journal

Medicine

20.3K 次观看.  Wake Forest University School of Medicine. RNA干扰（RNAi）拥有超过基因敲除的许多优点，在基因功能研究的工具已被广泛使用。基于DNA载体的RNAi技术的发明，已作出长期和诱导基因敲除可能，也增加了基因沉默的可行性在体内。

视频: DNA Vector-based RNA Interference to Study Gene Function in Cancer

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

在犬体内的肌肉功能测定

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

科研

医学

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

一个<em在体外系统来研究肿瘤休眠和转移性增长

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

原位膀胱癌模型的基因传递研究

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

无创性气管内插管，以研究小鼠肺的病理生理

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

三维文化：一种工具来研究正常腺泡结构与恶性转化的乳腺细胞

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

一个<em&gt;体外</em&gt;模型在乳腺癌研究激素的作用

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

在体内和体外途径研究银河系结构现货卵巢癌转移定植腹膜脂肪

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

使用Saccadometry与深部脑刺激来研究正常和病理脑功能

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...